JPH0283448A - Immunoassay - Google Patents

Abstract

PURPOSE:To simply perform the determination of the antigenic substance of every kind such as the drug or metabolite in body fluids by taking out a definite amount of a liquid phase after antigen-antibody reaction and measuring the enzymatic activity of the liquid phase using a dry chemical analysis film. CONSTITUTION:In a heterogenous system EIA (enzyme labelled immunoassay), a definite amount of a liquid containing an F-type marker is taken out after antigen-antibody reaction and the enzymatic activity thereof is measured using a dry chemical analysis film containing all of reagents necessary for measuring enzymatic activity to determine an antigenic substance of every kind by simple operation. As the enzyme used in labelling, for example, glucose oxidase (GOD) can be used and, by measuring F-type labelling quantity using an analytical film for measuring GOD activity, human ferritin of 10-600ng/ml can be determined and this calibration curve is used to make it possible to determine human ferritin in an arbitrary serum specimen.

Description

[Detailed Description of the Invention] (Field of Industrial Application) Analysis of drugs and metabolites contained in body fluids such as blood and urine is extremely useful for diagnosing pathological conditions and determining the course of treatment, and is an important part of clinical testing. occupies the field.

The present invention relates to methods for measuring these substances.

(Prior Art) Conventionally, many methods are known for detecting substances of biological origin. One large methodological group is immunoassays, which are divided into radioimmunoassays, enzyme immunoassays, fluorescence immunoassays, etc. depending on the type of labeling substance. Enzyme immunoassay (hereinafter referred to as EIA) is a method for quantifying antigens or antibodies by using enzyme activity as a marker in an antigen-antibody reaction system. The methods can be broadly divided into two. In other words, depending on whether it is necessary to separate antigen-antibody reaction products (bound type, B type) and non-reactants (free type, F type), homogeneous immunoassays (no separation required) and heterogeneous immunoassays ( (requires separation). These are described in detail in Eiji Ishikawa, edited by Enzyme Immunoassay Methods, 2nd edition, Igaku Shoin, 1982 (hereinafter referred to as Document (1)).

In heterogeneous immunoassays, the separation of type B and type F (
B/F separation) is required, and complicated and time-consuming operations are required, such as transferring the reaction product to a separate container, preparing reagents, adding substrate and reaction stop solution, and measuring solution absorbance. , which was extremely inefficient.

(Technical Issue B to be Solved) In order to solve these drawbacks, the present invention aims to improve the transfer of reaction products to separate containers, preparation of reagents, substrates and reactions in heterogeneous enzyme-labeled immunoassays. Eliminates the need for complicated and time-consuming operations such as adding a stop solution and measuring the absorbance of a solution.
The present invention provides an immunoassay method capable of quantifying various antigenic substances such as drugs and metabolites in body fluids.

(Means for Solving Technical Problems) The present invention is used in heterogeneous EIA (enzyme-labeled immunoassay) to remove a certain amount of a solution containing an F-type labeled substance after an antigen-antibody reaction and measure the enzyme activity. This immunoassay method is characterized in that various antigenic substances can be quantified with simple operations by measuring using a dry chemical analysis film containing all necessary reagents.

That is, the antibody or antigen is first physically or chemically bonded to a suitable porous membrane such as a cellulose acetate porous membrane made by a phase separation method, and the porous membrane obtains the insolubilized antibody or antigen. After performing an antigen-antibody reaction on this, a certain amount of the liquid phase (containing the free label) is taken out and its enzyme activity is measured using a dry chemical analysis film.

The antigen referred to herein is one that reacts with a specific antibody contained in a biological sample, and includes haptens and derivatives thereof.

Porous membranes that are known in the field of immunoanalysis can be used. For example, porous membranes made of cellulose nitrate, cellulose acetate, polyamide (e.g. nylon), polytetrafluoroethylene, etc.
These include agarose membrane, cellulose filter paper, glass fiber filter paper, etc. phase-inversion method
ocess: For example, Robert E.

by esting 5ynthetic Po1
y＋＊eric HebrainsMcGraw-
Preferred are cellulose nitrate porous membranes and cellulose acetate porous membranes made by the former If, New York, 1971;
Specification No. 67, especially from the bottom of page 347 of the publication, 34
You can use the ones listed at the top of page 8. Glass or plastic beads can be used instead of porous membranes, but antibodies insolubilized in beads are generally unstable in dry conditions and must be stored in a stabilizing solution containing serum albumin, etc. . On the other hand, antibodies insolubilized in a porous membrane can remain stable even in dry conditions. Porous membranes also have the advantage of being uniform and easy to mass-produce.

Dry chemical analysis films for measuring enzyme activity are disclosed in, for example, Japanese Patent Publication No. 53-21677 and Japanese Patent Application Laid-open No. 57-16-8.
No. 159, JP-A-62-175199, JP-A-62-
It can be produced using the description in No. 275699 and the like. The dry chemical analysis film may consist of one layer or multiple layers.

When the chemical analysis film consists of multiple layers, it is preferable that each layer is integrated, but it is preferable that the layers or groups of layers can be separated.
A chemical analysis film, which may be such that a liquid can temporarily flow between them, is a water-permeable film that detects the substrate of a reaction involving an enzyme (enzyme reaction) and the reagent that detects the reaction product of the enzyme reaction. Contained in the layer. Instead of the above reagents, a reagent that can quantify the reaction components of the enzymatic reaction may be included. Detection of the enzymatic reaction using the reagent is conveniently carried out optically using color development or color change of the reagent. However, it can also be carried out electrically or magnetically. The substrate may be a self-developing substrate, in which case the above-mentioned reagents can be omitted. Substrate and reagent may be contained in the same layer;
The integrated dry multilayer analysis film, which may be included in different layers, preferably has a spreading layer for spreading the sample liquid. Preferably, the liquid can be spread.

The integrated dry multilayer analytical film, in which at least one of the substrate and the reagent may be included in the developing layer, also preferably has a light-transparent support, although the light-transparent support may be water-permeable. A light-transparent and water-impermeable support, which is preferably water-impermeable, is provided on the surface opposite to the spreading layer.

In the method of the present invention, after the completion of the immune reaction, a certain amount of the liquid phase is spotted on a dry chemical analysis film for measuring enzyme activity, and changes in the reflected optical density (OD Measure using a concentration meter. The reaction rate is determined from the rate of change in optical density, the enzyme activity is calculated, and the concentration of the antigen in the sample is determined from the enzyme activity.

There are no particular restrictions on the enzymes used for drifting, and general EIA can be used.
Typical examples include glucose oxidase (GOD), peroxidase (POD), and flukaline phosphatase (ALP).
), β-galactosidase (β-GAL), and the like. Various enzyme-labeled antigens (or enzyme-labeled antibodies) can be easily obtained by referring to the above-mentioned document (1).

In the sandwich immunoassay according to the first aspect of the present invention, an antibody is first physically and/or chemically bonded to the surface of a suitable porous membrane.

After binding the antibody to the porous membrane, an aqueous solution of an immunologically inert substance (blocker) such as gelatin or serum albumin is added.
) to block non-specific binding sites on the surface of the porous membrane.
[6] containing a surfactant such as 0 is washed with a solution or saline to obtain a porous membrane in which the antibody is insolubilized.

A sample containing an antigenic substance (serum, plasma, urine, etc.) is added to the porous membrane in which the above-mentioned antibodies have been insolubilized, and then incubated at 0 to 37°C for several hours to overnight, preferably at 10 to 25°C for 2 hours.
After reacting for 10 to 10 hours, a certain amount of enzyme-labeled antibody is added to the plate, which is then washed with the above-mentioned washing solution. React for several hours to overnight at 0°C to 37°C, preferably for 2 to 10 hours at 10°C to 25°C. After the reaction is complete, a certain amount of the reaction solution is spotted on a dry chemical analysis film for enzyme activity measurement and reflected. By measuring the optical density (ODr), antigenic substances in a sample can be quantified. The amount of enzyme-labeled antibody should be slightly in excess of the antigenic material in the sample, but not excessively so.

In the immunoassay according to the second method of the present invention, the antigen is first physically and/or chemically bound to the surface of a suitable porous membrane. After binding the antigen to the porous membrane, a certain amount of an aqueous solution (blocker) of an immunologically inactive substance such as gelatin or serum albumin is added to block non-specific binding sites on the surface of the porous membrane. Next, the above porous membrane is washed with a buffer solution containing a surfactant such as Tween 20, saline, etc. to obtain a porous membrane in which the antigen is insolubilized.

A sample containing antibodies (serum, plasma, urine, etc.) is added to the above porous membrane in which the antigen has been insolubilized, and the mixture is heated for several hours to overnight at 0°C to 37°C, preferably for 2 to 10 hours at 10°C to 25°C. Make it react. Wash with the above-mentioned washing solution, add a certain amount of enzyme-labeled second antibody, and react at 0°C to 37°C for several hours to 1 day and night, preferably at 10°C to 25°C for 2 to 10 hours. 0 Reaction completed. Thereafter, the antibody in the sample can be quantified by spotting a certain amount of the reaction solution onto a dry chemical analysis film for measuring enzyme activity and measuring the reflected optical density (OD r ). The enzyme-labeled second antibody used in this method is selected to act as an antibody (anti-human globulin antibody) against the antigenic site of the antibody in the sample.

The amount of the enzyme-labeled second antibody should be slightly in excess of the antibody in the sample, but not excessively so.

The method of the present invention will be described in detail below with reference to Examples, but the present invention is not limited thereto.

[Example 1] (Measurement of human ferritin) 1”) CO
D Preparation of analytical film for activity measurement Transparent polyethylene terephthalate film (thickness 180 mm) whose surface has been made hydrophilic.
A coating solution having the following composition was coated on the sample at a ratio of 155 x 1/x'' and dried to form a coloring layer.

Round color layer coating liquid composition: Gelatin 160g water
1ooo g peroxidase 150,000 units Leuco dye (Structure below)
2.49bis[(vinylsulfonylmethyl
1.6g carbonyl) aminocomethane leuco dye: 2-(4-hydroxy-3,5-dimethoxyphenyl)
-4-(4-(dimethylamino)phenyl-5-phenethylimidazole) Next, apply a coating solution having the following composition on top of this at 62.5 xl/w.
” and dried to form an adhesive layer.

Adhesive layer coating liquid composition: Gelatin 60g water
11409 glucose
After moistening the above adhesive layer with 40 g of water at a concentration of about 30 g/I, a tricot knitted fabric made of polyethylene terephthalate spun yarn (36 gauge, 50 denier) was pressed and dried to obtain a spreading layer. A coating solution having the following composition was applied to the above development layer at a rate of 120 zl/m'' and dried.

Developing layer coating solution 5% (Ayu) Polyvinylpyrrolidone aqueous solution 200g Glucose 20 lN-2-hydroxyethyl 9.4g Piperazine-N' -
2-ethanesulfonic acid (adjusted to pn 6.5 with 2N-NaOH) An analytical film for measuring GOD activity was prepared by the above method.

2) Insolubilization of antibodies A solution of anti-human ferritin mouse antibody (1001000 AL!) obtained by immunizing mice according to a conventional method was added at 0.02 MIJ-M). Street liquid (pH 7.4, hereinafter PBS)
), and 2.5 μl of it was prepared using
Cellulose acetate porous membrane cut into mm discs (
Microfilter FM300 manufactured by Fuji Photo Film Co., Ltd.
) was added. After drying for 10 minutes, 1% human serum albumin was added to block non-specific binding sites on the porous membrane.
2.5 μm of BS was added and dried for 10 minutes. 0.05
%Tween20 (manufactured by Tokyo Kasei Kogyo Co., Ltd.)
It was washed with BS to obtain an antibody-insolubilized porous membrane.

3) Creation of a calibration curve Place each of these antibody-insolubilized porous membranes in a test tube,
Standard samples containing various amounts of human ferritin (PBS containing 0.1% human serum albumin) ranging from 600 ng/ml to 600 ng/ml were added. After incubating for 2 hours at room temperature, it was washed with PBS containing 0.05% Tween 20 (manufactured by Tokyo Kasei Kogyo Co., Ltd.), and then a GODtF-recognizing human ferritin antibody solution (COD 20 per 12
0111 units) was added and incubated for an additional 2 hours.

10 μl of the reaction solution in each test tube was taken out, and the free type (F type) GOD activity in the solution was measured using an analytical film for measuring GOD activity. That is, reaction solution 10 in each test tube
Using a micropipette, spot μl onto an analytical film for measuring GOD activity, and measure 1 to 5 minutes (+in.
) A calibration curve was created by measuring the change in reflected optical density (800 r). The results are shown in Figure 1.

Figure 1 shows that the amount of F-type labeling is measured using an analytical film for determining GOD activity.
This shows that it is possible to quantify human ferritin at 1/ml. Using this calibration curve, human ferritin in any serum sample could be quantified.

[Example 2] (Mumps virus IgG antibody measurement) 1'
) Preparation of analytical film for measuring GOD activity A film was prepared in exactly the same manner as in Example 1.

2) Antigen insolubilization Human ferritin in Example 1 An antigen insolubilized porous membrane was obtained in the same manner as in Example 1, except that mumps virus HA antigen (commercially available) was used instead of the mouse antibody.

That is, commercially available HA was added to the cellulose acetate porous membrane.
Antigen in carbonate buffer (salt concentration 0.05M, pH 9.6)
Add 2.5 μl of the solution diluted 10 times with Example 1.
I performed the same operation.

3) Creation of a calibration curve Using serially diluted mumps virus antibody positive serum as a sample, the same procedure as in Example 1 for creating a calibration curve was used to obtain changes in the absorbance of the analysis slide according to the serum dilution rate. . The results are shown in FIG. 2. Using this calibration curve, it was possible to detect mumps virus IgG antibodies in any serum sample.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the human ferritin assay obtained in Example 1. FIG. 2 is a graph showing the test line of the mumps virus IgG antibody obtained in Example 2. Applicant Fuji Photo Film Co., Ltd.

Claims (2)

[Claims] (1) In heterogeneous immunoassay using enzyme labeling,
A certain amount of antibody is physically and/or chemically bound to a porous membrane to obtain an insolubilized antibody, and after contacting the insolubilized antibody with the antigen in the sample, a certain amount of enzyme-labeled antibody is further contacted. A method for quantifying antigens in a sample, which is characterized by causing an antigen-antibody reaction by causing an antigen-antibody reaction, taking out a certain amount of the liquid phase after the antigen-antibody reaction, and measuring the enzyme activity of the liquid phase using a dry chemical analysis film. An immunoassay method for (2) In heterogeneous immunoassay using enzyme labeling,
A certain amount of antigen is physically and/or chemically bound to a porous membrane to obtain an insolubilized antigen, and after contacting the insolubilized antigen with an antibody in a sample, a certain amount of enzyme-labeled antibody is further contacted. A method for quantifying antibodies in a sample, which is characterized by causing an antigen-antibody reaction by causing an antigen-antibody reaction, taking out a certain amount of the liquid phase after the antigen-antibody reaction, and measuring the enzyme activity of the liquid phase using a dry chemical analysis film. An immunoassay method for